Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer

Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) contribute to tumour progression and metastasis. Assessment of transcription factors involved in these two mechanisms can help to identify new targets for an oncological therapy. In this study, we focused on the evaluation of the transcription factor Six1 (Sine oculis 1).

Lerbs et al BMC Cancer (2017) 17:249 DOI 10.1186/s12885-017-3225-5 RESEARCH ARTICLE Open Access Inhibition of Six1 affects tumour invasion and the expression of cancer stem cell markers in pancreatic cancer Tristan Lerbs1, Savita Bisht2, Sebastian Schölch3, Mathieu Pecqueux3, Glen Kristiansen4, Martin Schneider1, Bianca T Hofmann5, Thilo Welsch3, Christoph Reissfelder3, Nuh N Rahbari3, Johannes Fritzmann3, Peter Brossart2, Jürgen Weitz3, Georg Feldmann2† and Christoph Kahlert1,3*† Abstract Background: Epithelial-to-mesenchymal transition (EMT) and cancer stem cells (CSC) contribute to tumour progression and metastasis Assessment of transcription factors involved in these two mechanisms can help to identify new targets for an oncological therapy In this study, we focused on the evaluation of the transcription factor Six1 (Sine oculis 1) This protein is involved in embryologic development and its contribution to carcinogenesis has been described in several studies Methods: Immunohistochemistry against Six1 was performed on a tissue microarray containing specimens of primary pancreatic ductal adenocarcinomas (PDAC) of 139 patients Nuclear and cytoplasmic expression was evaluated and correlated to histopathological parameters Expression of Six1 was inhibited transiently by siRNA in Panc1 and BxPc3 cells and stably by shRNA in Panc1 cells Expression analysis of CDH1 and Vimentin mRNA was performed and cell motility was tested in a migration assay Panc1 cells transfected with Six1 shRNA or scrambled shRNA were injected subcutaneously into nude mice Tumour growth was observed for four weeks Afterwards, tumours were stained against Six1, CD24 and CD44 Results: Six1 was overexpressed in the cytoplasm and cellular nuclei in malignant tissues (p < 0.0001) No correlation to histopathological parameters could be detected Six1 down-regulation decreased pancreatic cancer cell motility in vitro CDH1 and vimentin expression was decreased after inhibition of the expression of Six1 Pancreatic tumours with impaired expression of Six1 showed significantly delayed growth and displayed loss of the CD24+/CD44+ phenotype Conclusion: We show that Six1 is overexpressed in human PDAC and that its inhibition results in a decreased tumour progression in vitro and in vivo Therefore, targeting Six1 might be a novel therapeutic approach in patients with pancreatic cancer Keywords: Six1, Pancreatic cancer, Epithelial-mesenchymal transition, Cancer stem cells * Correspondence: christoph.kahlert.079@googlemail.com † Equal contributors Department of General, Visceral and Transplantation Surgery, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Fetscherstr 74, 01307 Dresden, Germany Full list of author information is available at the end of the article © The Author(s) 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Lerbs et al BMC Cancer (2017) 17:249 Background Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumour with a poor prognosis Despite its low prevalence, it is the fourth leading cause of cancer-related death in western countries [1] Pancreatic cancer spreads rapidly and is highly resistant to chemotherapy These features are determined by several biological features, which are considered to be hallmarks of tumour development and dissemination [2] Among those fundamental cornerstones, epithelial-to-mesenchymal transition (EMT) plays a crucial role in tumour progression By adopting a more mesenchymal phenotype, cells increase motility, augment invasiveness und enhance chemoresistance [3] EMT is strongly connected to the concept of cancer stem cells (CSC) [4] In this model, CSC represent only a minor fraction of a tumour but are hypothesized to be crucially involved in its progression [5] They can divide infinitely and are strongly resistant to chemotherapeutics Therefore, they can survive chemotherapy and form recurrent disease [2] Brabletz et al described a model, in which migrating CSC are responsible for tumour dissemination whereas the epithelial non-CSC form is responsible for the growth of a single tumour [4] In good accordance with this assumption, Martin et al showed that EMT augments self-renewal capability [6] In this study we focused on the embryologic transcription factor Six1 (Sine Oculis 1) It contributes to organogenesis by inducing proliferation, migration and survival [7–9] In tumour biology, however, Six1 exerts protumourigenic functions by regulating EMT-related mechanisms [10] The role of Six1 in carcinogenesis has already been studied in several malignancies including breast cancer [11, 12], cervical cancer [13, 14] ovarian cancer and hepatocellular cancer [15, 16] Recently, our group has shown that overexpression of SIX1 is an independent prognostic marker in stage I - III colorectal cancer [17] Moreover, Li et al [18] and Jin et al showed an overexpression of Six1 in PDAC in their recent studies In the study by Jin et al Six1 was also an independent prognostic marker in pancreatic cancer [19] Additionally, Ono et al showed that Six1 promotes EMT by activating ZEB1 [20] The purpose of the present study was to evaluate the impact of Six1 expression on CSC- and EMT-phenotypes in PDAC To this end, we analysed a tissue microarray including 139 patients Furthermore, we assessed the impact of Six1 on EMT markers and migration in vitro in Panc1 and BxPc3 cells Finally, we investigated the impact of Six1 on tumour growth in vivo in a xenograft model Methods Patients The Medical Ethical Committees of the University of Bonn has approved the use of the patient tissue samples Page of 10 and clinic-pathological information in this study (Antragsnummer 13–091) Written informed consent was obtained from each patient prior to this study The study cohort included 139 patients who underwent tumour resection at the University Hospital of Bonn between 1998 and 2009 The analysis was performed retrospectively and it was not possible to deduce patient identity from patient data Cores derived from cancer tissue as well as from adjacent non-affected normal pancreatic parenchyma were analyzed Immunohistochemistry Immunohistochemical staining of human tissue microarray samples was performed as described previously [21] Likewise, immunohistochemical staining on whole tissue specimens from xenograft samples was conducted μm sections of formalin-fixed, paraffin-embedded tumour specimens were cut and mounted on SUPERFROST® PLUS microscope slides (Menzel, Germany) After overnight incubation at 37 °C, samples were dewaxed with xylol, rehydrated in a graded series of ethanol and subjected to heat-induced antigen retrieval (Dako REAL™ Target Retrieval Solution, pH 6.00, DAKO Denmark A/S) in a pressure cooker for 15 Nonspecific binding was blocked using an Avidin/Biotin Blocking Kit (Vector Laboratories, Inc., Burlingame, CA, USA) After antigen retrieval, slides were placed in an automated staining machine (DAKO Automatic Stainer) and incubated with the primary antibody for 30 Whole tissue specimens from xenografts specimens were additionally incubated with primary antibodies against CD44 (Rabbit monoclonal, ab151037, abcam, United Kingdom) and CD24 (Rabbit monoclonal, ab17982, Abcam, United Kingdom) for 30 Incubation with primary antibodies was followed by the biotinylated secondary antibody (DAKO REAL™ Biotinylated Secondary Antibody Anti -Rabbit, part of the DAKO REAL™ Detection System Peroxidase/AEC, Rabbit/Mouse, Code K5003, DAKO, Denmark) for 20 Afterwards, endogenous peroxidase was inhibited (DAKO REAL™ Peroxidase blocking solution, DAKO, Denmark) for followed by incubation with DAKO REAL™ streptavidin peroxidase (HRP) solution (part of DAKO REAL™ Detection System Peroxidase/AEC, Rabbit/Mouse, Code K5003, DAKO, Denmark) for 20 Finally, the specimens were visualised with DAKO REAL™ AEC/H2O2 Substrate Solution (part of DAKO REAL™ Detection System Peroxidase/ AEC, Rabbit/Mouse, Code K5003, DAKO, Denmark) and counterstained with haematoxylin Two independent researchers (CK and TL) estimated the expression of SIX1 on a blind basis A multi-head microscope was used and consensus was reached for each slide The staining intensity in cytoplasm was classified as absent: 0, weak or intermediate: and strong: For cell nucleus staining, the Lerbs et al BMC Cancer (2017) 17:249 percentage of positive cells was assessed: absent: 0, 0– 25%: 1, 25–50%: 2, 50–75%: 3, > 75%: Cell lines and transfection Panc1 and BxPc3 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA 20108, USA) Tumour cells were maintained in RPMI-1640 (Sigma, St Louis, MO), supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37 °C The anti-Six1 shRNA plasmid (Mission® shRNA bacterial glycerol stock, SHCLNGNM_005982, Sigma, USA) or an empty control vector (pLKO.1-puro, SHC001, Sigma, USA) were transfected using calcium phosphate-mediated transfection285 (ProFection® Mammalian, Cat No E1200, Promega, Germany) according to the manufacturer’s protocol Twenty-four hours after transfection, the cells were passaged 1:15 in appropriate medium containing μg/ml puromycin for puromycin selection Transfection efficiency was determined by quantitative RT-PCR (qPCR) A transient siRNA transfection [22] was performed using Lipofectamine 2000 [23] (invitrogen, USA) according to the manufacturer’s specifications An anti-Six1 siRNA from Sigma (Additional file 1: Table S1) and a negative control siRNA (AllStars, Qiagen, Netherlands) were purchased Per 1200 pmol of siRNA, 30 μl of Lipofectamine 2000 were used for transfection Afterwards, cells were incubated for 24 h and transfection efficiency was determined using quantitative RT-PCR (qPCR) RNA extraction and quantitative RT-PCR [24, 25] Total RNA from Panc1 and BxPc3 cells was extracted with the miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manual’s instructions RNA concentration was determined by a spectrophotometer (Nano Drop® 1000, Thermo Scientific, Germany) and reversely transcribed using the miScript Reverse Transcription Kit (Qiagen, Hilden, Germany) Five nanogram of the resulting cDNA was further subjected to qPCR (SYBR Green PCR Kit, Qiagen, Hilden, Germany) in a Roche Light Cycler™ (Roche Diagnostics GmbH, Mannheim, Germany) Ready specific primer pairs were purchased from Qiagen Samples were normalized to GAPDH RNA and fold change of expression was calculated according to the 2-ΔΔct method as previously described [26] Cell migration assay The migration assay was performed using 24 well migration chambers (ThinCerts™, μm pore, Greiner Bio-One, 1780 Wemmel, Belgium) Panc1 and BxPc3 cells were starved overnight Subsequently, 20.000 cells were plated in each migration chamber in 300 μl serumfree medium Subsequently, the migration chambers Page of 10 were placed on 24 well plates containing medium with 10% (v/v) fetal calf serum After an incubation for 24 h, Panc1 and BxPc3 cells at the bottom of the migration chamber were stained with 4′, 6-diamidino-2-phenylindole (DAPI) 20 representative figures of each migration membrane were taken using a fluorescent microscope and the number of migrated cells of each assay was counted All assays were performed in triplicates Xenograft model The study was approved by the regional authority for Nature, Environment and Consumer protection of the Land of North Rine-Westphalia (84–02.04.2015.A038) We used two groups each containing five mice (Athymic Nude Mouse, Crl:NU(NCr)-Foxn1nu, Charles River, VA, USA) 2.5 × 106 cells were injected in each flank Tumour growth and mice weight were assessed weekly for four weeks After four weeks, the mice were euthanasized Tumour samples were fixed in formalin and embedded in paraffin for further immunohistochemical analyses Statistical analysis The software package GraphPad Prism, version (GraphPad Software, La Jolla, CA, USA) was used for all calculations Pearson’s r test was applied to analyze the correlation between the expression of Six1 and pathological parameters Differences in expression of Six1 in the PDAC cohort, Panc1 and BxPc3 cells, differences in migration and differences in tumour growth in vivo were assessed using the Student’s t-test The p values of all statistical tests were 2-sided, and p ≤ 0.05 was considered to indicate a statistically significant result Results Expression of Six1 in pancreatic ductal adenocarcinoma and its histopathological correlation Patient characteristics and clinical specimens Tissue samples from 139 patients suffering from primary pancreatic cancer were evaluated by IHC, out of these, sufficient material and data for final analysis were available in 137 cases Of those 137 patients the median age was 66 years (36–85) 74 patients were male, 59 female The UICC tumour stage at time of tumour resection was I in cases, II in cases, III in 123 cases and IV in cases 98 patients had positive lymph node metastasis (pN1), 38 patients were free of lymph node metastasis (pN0) and in patient lymph node status was not known Tumour grading was I in case, II in 59 cases and III in 54 cases In 23 cases grading could not be exactly determined Characteristics of the cohort are shown in Table Lerbs et al BMC Cancer (2017) 17:249 Page of 10 Table Correlation of Six1 expression in cytoplasm to histopathological parameters Parameter Six1 expression in malignant tissue Six1 expression in benign tissue Number No Weak Strong 137 50 (36,5%) 66 (48,2%) 21 (15,3%) < Median 68 (49,6%) 26 (34,8%) 31 11 ≥ Median 69 (50,4%) 24 (34,8%) 35 (50,7%) 10 (14,5%) Total p-value Number No Weak Strong p-value 105 91 (86,7%) 14 (13,3%)